Recently, a process for synthesizing a compound using a biocatalyst has been used for producing a variety of compounds because of advantages such as moderate reaction conditions, simplified reaction processes, and high purity of reaction products due to small amounts of by-products.
Since the discovery of nitrile hydratase, an enzyme that converts a nitrile compound into an amide compound, biocatalyst utilization has been actively studied in the production of amide compounds (JP Patent Publication (Kokai) No. 11-123098, JP Patent Publication (Kokai) No. 7-265091, JP Patent Publication (Kokoku) No. 56-38118, and JP Patent Publication (Kokai) No. 11-89575).
At present, a microorganism having nitrile hydratase activity is used for producing acrylamide, nicotineamide, or the like at the industrial level for a superior reaction process from the viewpoints of operability, safety, economic efficiency, and other factors.
Up to the present, a considerable number of microorganisms have been found having nitrile hydratase activities. Examples thereof include microorganisms belonging to the genera Nocardia, Corynebacterium, Bacillus, Pseudomonas, Micrococcus, Rhodococcus, Acinetobacter, Xanthobacter, Streptomyces, Rhizobium, Klebsiella, Enterobacter, Erwinia, Aeromonas, Citrobacter, Achromobacter, Agrobacterium, and Pseudonocardia. 
Among them, the genera Pseudomonas, Bacillus, Rhodococcus, and Pseudonocardia express a nitrile hydratase having a very high level of activity and stability. Thus, they are used at the industrial level or levels similar thereto.
Further, when culturing these microorganisms, it is known that a microbial cell having highly active nitrile hydratase is obtained by a method of adding nitriles or amides (JP Patent Publication (Kokoku) Nos. 61-43996 and 61-43999), a method of adding amino acid (JP Patent Publication (Kokoku) Nos. 61-43997 and 61-43998), a method of adding some kind of metal ion (JP Patent Publication (Kokai) No. 61-162193, JP Patent Publication (Kokoku) No. 6-55148, and JP Patent Publication (Kokai) No. 8-187092), or the like.
In contrast, a biocatalyst has low stability with regard to heat, and thus, reactions must be carried out at low temperatures. This results in a decreased reaction speed per catalyst. When producing a compound using a biocatalyst at the industrial level, therefore, the catalyst concentration in the reaction tank should be raised.
A currently known industrial process for producing an amide compound from a nitrile compound using a biocatalyst is similarly carried out by immobilizing microbial cells to make them particulate, raising the catalyst concentration in the reaction tank, and facilitating catalyst separation (see Kagaku to Kogyo (Chemistry and Chemical Industry) Vol. 43, No. 7, p. 1098-1101 (1990), JP Patent Publication (Kokai) Nos. 54-143593 and 54-144889). Also, a method for immobilizing the cell is studied (see JP Patent Publication (Kokai) Nos. 57-39792 and 62-294083). When effective production of an amide compound at the industrial level is intended, immobilization of cells at a higher concentration has been considered to be important (see JP Patent Publication (Kokai) No. 7-203964).
However, the present inventors entrap-immobilized a microbial cell that exhibits high nitrile hydratase activity and used it in the reaction. As a result, it was confirmed that a nitrile compound as a reaction substrate and/or an amide compound as a reaction product caused diffusion defects in the entrap-immobilized particles, and the reaction speed was significantly decreased.
For example, according to the comparison of initial reaction speeds between the entrap-immobilized cell and the unimmobilized cell, the reaction speed was significantly decreased to one-tenth or lower, depending on reaction conditions. Not only the initial reaction speed is significantly decreased, but also the activity of the enzyme in the entrap-immobilized catalyst, which does not fully contribute to the reaction due to diffusion defect, is lowered during the reaction. This also lowers the amount of amide compound produced per unit cell amount.
Specifically, decreased reaction speed or lowered amount of amide compound produced per unit cell amount as mentioned above results in unfavorable conditions. Under such conditions, it takes a long time to accumulate the targeted amount when producing an amide compound by a batch reaction, and the size of facilities must be enlarged in the case of a continuous reaction.
Accordingly, an object of the present invention is to solve problems occurring in the process for producing an amide compound from a nitrile compound using a biocatalyst, such as decreased reaction speed or lowered amount of amide compound produced per unit cell amount. These problems are caused by the use of immobilized microbial cell with highly exhibited nitrile hydratase activity.
In order to attain the above object, the present inventors have conducted concentrated studies concerning a more suitable form of catalyst than the immobilized catalyst. As a result, they have found that an amide compound could be more effectively produced when using a microbial cell that exhibits high nitrile hydratase activity of 50 U or higher per mg of dry cell at 10° C., and bringing the microbial cell into contact with a nitrite compound while suspended in an aqueous medium, than is the case when immobilizing the cell. This has led to the completion of the present invention.
More specifically, the present invention relates to a process for producing an amide compound from a nitrile compound using a microbial catalyst, wherein a microbial cell having nitrile hydratase activity of 50 U or higher per mg of dry cell at a reaction temperature of 10° C. is brought into contact with a nitrile compound in an aqueous medium without being immobilized.